None of the following discussion of the background of the invention is admitted to be prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine phosphatases (TPs) and tyrosine kinases (TKs), including receptor tyrosine kinases and non-receptor tyrosine kinases.
Receptor tyrosine kinases (RTKs) belong to a family of transmembrane proteins and have been implicated in cellular signaling pathways. The predominant biological activity of some RTKs is the stimulation of cell growth and proliferation, while other RTKs are involved in arresting growth and promoting differentiation. In some instances, a single tyrosine kinase can inhibit, or stimulate, cell proliferation depending on the cellular environment in which it is expressed.
RTKs are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric changes that activate the intracellular kinase domains and result in the self-phosphorylation (autophosphorylation and/or transphosphorylation) of the receptor on tyrosine residues. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signaling molecules, thereby activating various signal transduction pathways.
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 domains and SH3 domains. The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction.
A central feature of signal transduction is the reversible phosphorylation of certain proteins. Receptor phosphorylation stimulates a physical association of the activated receptor with target molecules, which either are or are not phosphorylated. Some of the target molecules such as phospholipase Cγ are in turn phosphorylated and activated. Such phosphorylation transmits a signal to the cytoplasm. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal transducer proteins. For example, receptor phosphorylation and the subsequent allosteric changes in the receptor recruit the Grb-2/SOS complex to the catalytic domain of the receptor where its proximity to the membrane allows it to activate ras. The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, Science, 254:1146-1153, 1991; Schlessinger, Trends Biochem. Sci., 13:443-447, 1988; and Ullrich and Schlessinger, Cell, 61:203-212, 1990.
A variety of mitogens as well as tumor promoters and agents which cause cellular differentiation can initiate a signalling cascade leading to phosphorylation and activation of mitogen-activated protein (MAP) kinases (also called ERKs). Boulton et al., Biochemistry 30:278-286 (1991) and Boulton et al., Science 249:64-65 (1990) describe the purification and cloning of a MAP2/MBP kinase which they named extracellular signal-regulated kinase 1 (ERK-1). Using probes derived from ERK-1, two novel kinases were identified, ERK-2 and ERK-3 (Boulton and Cobb, Cell Regulation 2:357-371, 1991; Boulton et al., Cell 65:663-675, 1991). A fourth ERK has been briefly described (Cobb et al., Cell Regulation 2:965-978, 1991 and WO 91/19008 published Dec. 12, 1991). These proline-site-directed serine-threonine kinases in turn phosphorylate transcription factors such as p65TCF/Elk-1, c-jun and c-myc and thus appear to play a crucial role in signal transduction by converting extracellular stimuli into transcriptional activation.
The mechanism of activation of MAP kinases has been intensively investigated and revealed a conserved signalling cascade initiated by ligand induced activation of receptor tyrosine kinases which leads to a sequential activation of a series of protein kinases. The activated growth factor receptor signals via ras to the serine-threonine kinase raf which directly activates the MAP-kinase-ERK-kinase MEK. MAP kinases are stimulated by phophorylation on two regulatory threonine and tyrosine residues, respectively, which is catalyzed by activated MEK. Upon activation, MAP kinases have been reported to translocate into the nucleus and phosphorylate transcription factors. Another substrate of MAP kinases is the S6 kinase II (pp90rsk) which is activated by ERKs and might then control protein translation. MAP kinases can also be activated by TPA which stimulates PKC and signals to MEK via raf. However, this pathway appears to be dependent on ras. A negative regulator of MAP kinase activity has been identified by cloning of a dual specificity phosphatase encoded by an immediate early gene (Charles et al. 1993, PNAS 90, 5292-5296) that seems to be highly specific for MAP kinases (Sun et al. 1993, Cell 75, 487-493).
One or more signal transduction pathways are belleived to control the differentiation of myoblasts. Upon depletion of serum growth factors from the culture medium at high cell density, proliferating skeletal myoblasts cease DNA synthesis, start to express muscle-specific genes (biochemical differentiation) and fuse to form multinucleate myotubes (terminal differentiation). However, upon terminal differentiation, myoblasts loose their ability to reenter the cell cycle in response to growth factor stimulation. This presumably involves the retinoblastoma gene product pRB and its interaction with members of the basic helix-loop-helix myogenic factor family.
While some of these muscle specific transcription factors, namely MyoD and myf5, are constitutively expressed both in cycling myoblasts as well as in myotubes, myogenin expression is induced when myoblasts start to differentiate. However, not only transcriptional regulation, but also posttranslational modification such as phosphorylation which has been reported for MyoD1 and myogenin as well as myf5 may influence commitment to myogenesis and maintenance of the differentiated state. Activated oncogenes like ras and src as well as growth factors which are involved in or initiate signal transduction, inhibit myogenesis. In addition, PKC is able to phosphorylate myogenin and could be a major mediator of this inhibition.